Mucolipins constitute a family of cation channels with homology to the transient receptor potential superfamily. In mammals, the mucolipin family includes three members (MCOLN1-3). MCOLN1 is the best-characterized member of the mucolipin family as mutations in this protein have been associated with mucolipidosis type IV (MLIV). MLIV is an autosomal recessive disease characterized by severe mental and psychomotor retardation, diminished muscle tone or hypotonia, achlorhydria, and visual problems including corneal clouding, retinal degeneration, sensitivity to light, and strabismus. MLIV is relatively frequent among Ashkenazi Jews, with 1/100 of the population estimated to be a genetic carrier. Two founder mutations that result in the absence of MCOLN1 account for 95% of the cases in this group. MCOLN1 is a 580-amino acid protein and has a molecular mass of 65 kDa. The predicted topology of the protein consists of six transmembrane-spanning domains with the N- and C- terminal tails oriented within the cytosol. To gain information on the function of MCOLN1, we addressed its intracellular distributions and trafficking. We have previously described that MCOLN1 localized to late endosomes and lysosomes. Two di-leucine motifs cooperate to regulate delivery of MCOLN1 to lysosomes through interactions with the clathrin adaptors AP1, AP2, and AP3. The C-terminal tail of MCOLN1 undergoes post-translational modifications that regulate its activity and trafficking. For example, palmitoylation of three cysteine residues (Cys565, Cys566, and Cys567) increases the rate of MCOLN1 internalization from the plasma membrane. In addition, PKA-mediated phosphorylation of Ser557 and Ser559 negatively regulates MCOLN1 channel activity in vivo.[unreadable] To a cellular level, MLIV is characterized by accumulation of enlarged vacuolar structures containing phospholipids, sphingolipids, mucopolysaccharides, and gangliosides. This accumulation of undigested material in lysosomes led to the suggestion that MCOLN1 plays a role in organelle biogenesis or regulation of lysosomal hydrolysis. However, the mechanism by which defects in MCOLN1 function result in mental and psychomotor retardation remains largely unknown. Recent studies reveal an interesting connection between lysosomal function, autophagy, and neurodegeneration. Autophagy is a crucial clearance mechanism that protects against the accumulation of toxic protein aggregates and damaged organelles. The last step of autophagy requires fusion of autophagosomes with late endosomes/lysosomes to ensure degradation of the autophagosomes content. [unreadable] We have examined the autophagic process in fibroblasts from MLIV patients. We reasoned that the defects on the late endosomal/lysosomal pathway observed in MLIV might have a profound impact on the formation and/or degradation of autophagosomes. Using multiple approaches, including LC3 localization, LC3-II/LC3-I westernblot, electron microscopy, and LC3/CD63 co-localization analysis, we find increased basal autophagy in MLIV fibroblasts. Autophagosome accumulation in MCOLN1-deficient cells is due to decreased autophagosome degradation and increased autophagosome formation. We also find increased levels of p62, a protein commonly found in protein inclusions associated with neurodegenerative disorders, as well as accumulation of ubiquitinated aggregates. Importantly, the majority of p62 accumulated in MLIV cells is resistant to extraction with detergents, an indication that p62 is part of aggregates or protein inclusions. [unreadable] Based on these data, we propose a model in which trafficking defects along the endosomal/lysosomal pathway due to absence of MCOLN1 impairs lysosomal function and results in inefficient autophagosome degradation. This in turn, causes accumulation of ubiquitinated aggregates that generate cellular stress and further induces autophagosome formation and accumulation. Defects in autophagy may not be especially detrimental in fibroblasts, where the rapid division of the cells helps to prevent the accumulation of misfolded or aggregated proteins. However, the accumulation of these products in neurons may result in cell death and, as the neurons cannot be replaced, neurodegeneration occurs. In addition, autophagic dysfunction also causes accumulation of damaged mitochondria. These abnormal mitochondria are deficient in ATP production, produce increased amounts of reactive oxygen species, and increase susceptibility of cells to pro-apoptotic stimuli.[unreadable] In summary, our data demonstrates the impact of lysosomal function on autophagic activity and suggest that, analogous to other lysosomal storage disorders, accumulation of ubiquitinated protein inclusions due to defective autophagy may contribute to the neurodegeneration observed in MLIV patients.[unreadable] MCOLN3 is another member of the mucolipin family that might play a role in different human pathologies. Mutations in MCOLN3 are responsible for the varitint-waddler mouse phenotype characterized by defects in pigmentation and hearing loss. MCOLN3 is located in hair cells, and it has been suggested that it may be implicated in hair cell maturation and melanosome trafficking. Several recent reports showed that TRPML3 is an inwardly (from lumen into cytoplasm) rectifying monovalent cation channel that is permeable to Ca2+ and suppressed by low pH. To better understand the function of MCOLN3 we decided to study its subcellular localization in the retinal epithelial cell line ARPE19. Using immunofluorescence and confocal microscopy we found that heterologously expressed GFP-tagged-MCOLN3 (GFP-MCOLN3) localized to the plasma membrane and to the endosomal pathway in ARPE19 cells. Overexpressed GFP-MCOLN3 showed extensive co-localization with the early endosomal markers Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), while it co-localized to less extent with late endosomal markers such as LBPA (lysobisphosphatidic acid). These results show a clear difference in the subcellular distribution between MCOLN3 and its relative MCOLN1, which is mostly lysosomal. The localization of MCOLN3 within the upper, less acidic, portions of the endocytic pathway is consistent with the reported inhibition of the channel at low pH characteristic of lysosomes. Ongoing experiments will help us to determine the function of MCOLN3 within the endocytic pathway.